In the quantitative determination of total cholesterol, it is necessary to convert the cholesterol esters into free cholesterol. This conversion can be accomplished chemically by alkaline hydrolysis or enzymatically using cholesterol esterase as described in British patent specification No. 1,429,526.
British patent specification No. 1,429,526 suggests that a preferred cholesterol esterase for this purpose is that found in enzyme preparations obtained by growing Candida rugosa. Other useful sources for cholesterol esterases described in this British Patent include: Actinomyces aurcoverticillium WS 90002, Actinomyces cyaneofuscatus WS 90003, Actinomyces griseomycini WS 90004, Actinomyces longisporus-fl. WS 90005, Actinomyces malachiticus WS 90006, Actinomyces roseolus WS 90007, Actinomyces toxytricini WS 90008, Actinomyces variabilis Streptomyces spec. WS 90010, Streptomyces autotrophicus WS 90011, Streptomyces canescens WS 90012, Streptomyces chartreusis WS 90013, Streptomyces michiganensis WS 90014, Streptomyces murinus WS 90015, Streptomyces hachijoensis WS 90016, Streptomyces caelestes WS 90017, Streptomyces tendac WS 90018, Nocardia rubra WS 90019, Candida mycoderma WS 90020, Candida albicans WS 90021, Candida albicans WS 90022, Candida albicans WS 90023, Candida spec. WS 90024, Cunninghamella elegans WS 90025, Mucor muccdo WS 90026, Rhizopus spec. WS 90027, Penicillium spec. WS 90028 and Aspergillus spec. WS 90029.
British patent specification No. 1,429,526 states that most microbial cholesterol esterases, such as those from the microorganisms listed above, are bound in lipoid membranes and are difficult to purify. The enzyme preparation obtained from Candida rugosa generally has large amounts of lipase activity associated with its cholesterol esterase activity. A method for purifying enzyme preparations suggested by British patent specification No. 1,429,526 to achieve up to a 20 to 30 fold enrichment of cholesterol esterase involves dialysis with a weakly basic anion exchange and an ammonium sulfate fractionation. Such methods have not been successful in separating the lipase activity from the cholesterol esterase activity of enzyme preparations such as that obtained from Candida rugosa.
A dry multilayer element for the quantitative analysis of analytes such as total cholesterol has been proposed by Goodhue et al in French Pat. No. 2,266,170, published Oct. 24, 1975. When making such elements it is desirable to reduce the amount of unnecessary solids such as extraneous proteins in the coatings in order to produce stable uniform coatings. Thus, when using an enzyme preparation from Candida rugosa, for example, for its cholesterol esterase (i.e., ester hydrolase) activity, it is desirable to reduce the substantial amounts of unwanted and unnecessary lipase activity and the protein associated therewith. The lipase activity may also affect the stability of the interactive materials present in the element and the precision of the analysis. The present invention provides a method for substantially removing the separable lipase activity from the desired cholesterol esterase activity in an enzyme preparation having separable lipase and cholesterol esterase activities.